Method of transluminal application of myogenic cells for repair or replacement of heart tissue

ABSTRACT

A process is described for cellular repair of failing tissue of an organ if a patient&#39;s body. In the process, a catheter is positioned to provide an entry point for stem cell injection at the proximal end of the central lumen of the catheter external to the body and an exit point from the central lumen at the distal end proximate the site of the failing tissue. Stem cells are then injected through the catheter to invade the failing tissue at the site, while local forces at the site are quelled from disrupting migration of the stem cells into the failing tissue, to enhance the concentration of the stem cells and the pressure arising from the injection at the site and thereby overcome any barrier between the site of the injection and the failing tissue. Preferably, autologous adult stem cells are used in the procedure.

BACKGROUND OF THE INVENTION

The present invention relates generally to transluminal application ofmyogenic cells for tissue repair, such as myocardial repair, and moreparticularly to balloon catheter protected transluminal application ofmyogenic cells for repair of a failing body organ such as heart, brain,liver, kidney or pancreas. It is a principal aim of the invention toprovide a novel method to repair failing tissue.

In principle, the human body has three types of cells. One typeconstitutes cells that continuously undergo replication andreproduction, such as dermal cells and epithelial cells of theintestine, for example. These cells, which have a life as short as tendays, are replaced by the same cell type which is replicatingcontinuously. A second type of cell is differentiated in the adultstate, but has the potential to undergo replication and the ability toreenter the cell cycle under certain conditions, an example being livercells. The liver has the capacity to regrow and repair itself even if atumor is excised and a major portion of the liver is removed. The thirdcell type comprises those cells that stop dividing after they havereached their adult stage, such as neuro cells and myocardial cells.

For the latter type or group of cells, the number of cells in the bodyis determined shortly after birth. For example, myocardial cells stopdividing at about day ten after delivery, and for the rest of its lifethe human body has a fixed number of myocardial cells. Changes inmyocardial function occur not by division and new cell growth, but onlyas a result of hypertrophy of the cells.

Although the absence of cell division in myocardial cells is beneficialto prevent the occurrence of tumors—which practically never occur in theheart—it is detrimental with regard to local repair capacities. Duringthe individual's lifetime, myocardial cells are subjected to variouscauses of damage, that irreversibly lead to cell necrosis or apoptosis.

The primary reason for cell death in the myocardium is ischemic heartdisease—in which the blood supply to the constantly beating heart iscompromised through either arteriosclerotic build-up or acute occlusionof a vessel following a thrombus formation, generally characterized asmyocardial infarction (MI). The ischemic tolerance of myocardial cellsfollowing the shut-off of the blood supply is in a range of three to sixhours. After this time the overwhelming majority of cells undergo celldeath and are replaced by scar tissue.

Myocardial ischemia or infarction leads to irreversible loss offunctional cardiac tissue with possible deterioration of pump functionand death of the individual. It remains the leading cause of death incivilized countries. Occlusion of a coronary vessel leads tointerruption of the blood supply of the dependent capillary system.After some 3 to 6 hours without nutrition and oxygen, cardiomyocytes dieand undergo necrosis. An inflammation of the surrounding tissue occurswith invasion of inflammatory cells and phagocytosis of cell debris. Afibrotic scarring occurs, and the former contribution of this part ofthe heart to the contractile force is lost. The only way for the cardiacmuscle to compensate for this kind of tissue loss is hypertrophy of theremaining cardiomyocytes (accumulation of cellular protein andcontractile elements inside the cell), since the ability to replace deadheart tissue by means of hyperplasia (cell division of cardiomyocyteswith formation of new cells) is lost shortly after the birth of mammals.

Other means of myocardial cell alteration are the so-calledcardiomyopathies, which represent various different influences of damageto myocardial cells. Endocrine, metabolic (alcohol) or infectious (virusmyocarditis) agents lead to cell death, with a consequently reducedmyocardial function. The group of patients that suffer myocardial damagefollowing cytostatic treatment for cancers such as breast orgastrointestinal or bone marrow cancers is increasing as well,attributable to cell necrosis and apoptosis from the cytostatic agents.

Heretofore, the only means for repair has been to provide an optimalperfusion through the coronary arteries using either interventionalcardiology—such as PTCA (percutaneous transluminal coronaryangioplasty), balloon angioplasty or stent implantation—or surgicalrevascularization with bypass operation. Stunned and hibernatingmyocardial cells, i.e., cells that survive on a low energy level but arenot contributing to the myocardial pumping function, may recover. Butfor those cells which are already dead, no recovery has been achieved.

The current state of interventional cardiology is one of high standard.Progress in balloon material guide wires, guiding catheters and theinterventional cardiologist's experience as well as the use ofconcomitant medication such as inhibition of platelet function, hasgreatly improved the everyday practice of cardiology. Nevertheless, anacute myocardial infarction remains an event that, even with optimaltreatment today, leads to a loss of from 25 to 100% of the area atrisk—i.e., the myocardium dependent on blood supply via the vessel thatis blocked by an acute thrombus formation; A complete re-canalization byinterventional means is feasible, but the ischemic tolerance of themyocardium is the limiting factor.

A recent article published in the New England Journal of Medicine(Schömig A. et al., “Coronary stenting plus platelet glycoproteinIIb/IIIa blockade compared with tissue plasminogen activator in acutemyocardial infarction,” N Engl J Med 2000; 343:385-391), for which theapplicant herein was the primary clinical investigator, reports on astudy of the myocardial salvage following re-canalization in patientswith an acute myocardial infarction.

The average time until admission to the hospital in these patients was2.5 hours and complete re-canalization was feasible after 215 minutes,roughly 3.5 hours. Nevertheless, only 57% of the myocardium at riskcould be salvaged by re-canalization through interventional cardiologyby means of a balloon and stent. When the group of patients wasrandomized to the classical thrombolytic therapy, which is the worldwidestandard (with no interventional means), only 26% of the myocardium atrisk could be salvaged. This means that even under optimal circumstancesmore than 40% of the myocardial cells are irreversibly lost.

With the knowledge that many patients arrive at a hospital at from 6 to72 hours after the acute symptoms of vessel blockage by a thrombus, onecan assume that the average loss of affected myocardial tissue is in arange of from 75 to 90% following an acute MI.

As noted above, cells can survive on a lower energy level, referred toas hibernating and stunning myocardium. As the collateral blood flowincreases or re-canalization provides new blood supply they can recovertheir contractile function. The principle of myocardial re-perfusion,limitation of infarct size, reduction of left ventricular dysfunctionand their effect on survival were described by Braunwald (Braunwald E.et al., “Myocardial reperfusion, limitation of infarct size, reductionof left ventricular dysfunction, and improved survival: should theparadigm be expanded?,” Circulation 1989; 79:441-4).

Annually, about five million Americans survive an acute myocardialinfarction. Clearly then, loss of affected myocardial tissue is aproblem of major clinical importance. Currently, repair is limited tohypertrophy of the remaining myocardium, and optimal medical treatmentby a reduction in pre- and after-load as well as the optimal treatmentof the ischemic balance by β-blockers, nitrates, calcium antagonist, andACE inhibitors.

If it were feasible to replace the dead myocardium (scar tissue) byregrowing cells, such a technique would have a profound impact on thequality of life of affected patients.

As noted earlier herein, in addition to ischemic heart disease otherreasons exist for the reduction of myocardial cells that contribute tothe pumping function of the heart. Among them are the cardiomyopathies,which describe a certain dysfunction of the heart. Reasons are many,such as chronic hypertension which ultimately leads to a loss ineffective pumping cells, and chronic toxic noxious such as alcohol abuseor myocarditis primarily following a viral infection. Also, cell damagein conjunction with cytostatic drug treatment is becoming of greaterclinical relevance.

The group of Willam C. Claycomb et al. has been engaged in research onthe behavior and the development of myocytes since the early 1970's. Intheir initial report (Goldstein M. A. et al., “DNA synthesis and mitosisin well-differentiated mammalian cardiocytes,” Science 1974; 183:212-3),they described the incorporation of 3H-Thymidin into the nuclei of heartcells of two days old rats which indicates that neonatal cardiac cellsstill undergo synthesis of DNA and divide despite the presence ofcontractile proteins. This phenomenon of cell division ceases at day 17of the postnatal development. After that time no further division ofcardiac cells occurs, either in rats or in humans.

The interest in mammalian cardiomyocytes has led to the development ofcultures of adult cardiac muscle cells (Claycomb W. C. et al., “Cultureof the terminally differentiated adult cardiac muscle cell: A light andscanning electron microscope study,” Dev Biol 1980;80:466-482), andultimately to the generation of a transplantable cardiac tumor-derivedtransgenic AT1-cell.

During the 1980's intensive studies were conducted with thecharacterization of this atrial derived myocyte cell line, which isimmortalized by the introduction of the SV40-large-T-oncogene (SV40-T).From this AT-1-cell-group, other adult cardiomyocytes have been derived.These can be passaged indefinitely in culture, can be recovered from afrozen stock, can retain a differentiated cardiomyocyte phenotype, andmaintain their contractile activity. They are described as HL-1-cells.The reader is referred, for example, to Delcarpio J. B. et al.,“Morphological characterization of cardiomyocytes isolated from atrans-plantable cardiac tumor derived from transgenic mouse atria (AT-1cells),” Circ Res 1991; 69(6):1591-1600; Lanson Jr. N. A. et al., “Geneexpression and atrial natriuretic factor processing and secretion incultured AT-1 cardiac myocytes,” Circulation 1992; 85(5):1835-1841;Kline R. P. et al., “Spontaneous activity in transgenic mouse heart:Comparison of primary atria tumor with cultured AT-1 atrial myocytes,” JCardiovasc Electrophysiol 1993; 4(6):642-660; Borisov A. B. et al.,“Proliferative potential and differentiated characteristics of culturedcardiac muscle cells expressing the SV 40 T oncogene,” Card Growth Reg1995; 752:80-91; and Claycomb W. C. et al., “HL-1 cells: A cardiacmuscle cell line that contracts and retains phenotypic characteristicsof the adult cardiomyocyte,” Proc Natl Acad Sci USA 1998; 95:2979-84.

Finally, the cardiomyocyte transplantation in a porcine myocardialinfarction model has been studied intensively in collaboration with theresearch group of Frank Smart (Watanabe E. et al., “Cardiomyocytetransplantation in a porcine myocardial infarction model,” CellTransplant 1998; 7(3):239-246). In conjunction with the AT-1cardiomyocytes, human fetal cardiomyocytes were injected into the adultpig heart infarction area.

In summary, these cells showed local growth and survived in theinfarction border zone, but could not be found in the core scar tissueof the myocardial infarction. The majority of the implanted cells werereplaced with inflammatory cells, suggesting that the immuno-suppressantregimen that was concomitantly applied was not sufficient for thegrafted cells to survive in the host myocardium. Other factors that mayhave influenced the result that the transplanted cells were notdetected, could possibly be linked to the fact that the cells weregrafted 45 days after inducing the infarction.

It is known that the inflammatory stimuli for cell growth aresignificantly reduced in the first two to three weeks of an MI. Also,that transforming-growth-factor-b (TGF-b), fibroblast-growth-factor-2(FGF-2), platelet-derived-growth-factor (PDGF) and other cytokines, likethe interleucin-family, tumor-necrosis-factor-a (TNF-a) and interferon-gare strong stimulators of cell proliferation and cell growth. Theadjunct therapy with immuno-suppression has further reduced this stimulifor cell growth.

Another major factor for the failure of detection of grafted cells inthe myocardial scar may be the selection of the infarction model. Anartery is occluded and the blood supply has not recovered beforegrafting. There is no reason to assume that the grafted cells couldsurvive in an ischemic area and grow, better than the myocytes.

Therefore, other groups have tried to induce a myocardial angiogenesisby gene-therapy. This was either performed by the administration byfibroblast growth factor II in the presence or absence of heparin (seeWatanabe E. et al., “Effect of basic fibroblast growth factor onangiogenesis in the infarcted porcine heart,” Basic Res Cardiol 1998;93:30-7) or by application of vascular endothelial growth factor (VEGF),a potent mitogen for endothelial cells. VEGF stimulates capillaryformation and increases vascular permeability (Lee J. S. et al., “Genetherapy for therapeutic myocardial angiogenesis: A promising synthesisof two emerging technologies,” Nat Med 1998; 4(6):739-42). Still othergroups have tried to increase the collateral capillary blood flow byhuman bone marrow derived angioblasts and have shown an improvement inacute myocardial infarction in rats treated with injections ofcolony-stimulating-factor-G (CSF-G) mobilized adult human CD-34 cells(Kocher A. A. et al., “Neovascularization of ischemic myocardium byhuman bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis,reduces re-modeling and improves cardiac function,” Nat Med 2001;7(4):430-6).

While these approaches certainly have some research merit, theirclinical relevance for the majority of patients is not as important,since we have effective means to re-canalize an occluded vessel andprovide a blood supply via the natural branching of the coronaryarteries, which further subdivides into arterioles and capillaries.

Other attempts to transplant preformed patches also necessitate thegrowth of the grafted cells in a patch formation and a surgicaloperation in a patient, which requires opening the thoracic cage.

Considering the complications, the cost and the risk associated withthese time consuming procedures, it becomes clear that they offer onlylimited likelihood for widespread routine application.

Other groups have tried to make use of the precursor cells that arefound in the peripheral muscle. Unlike the heart, there is a certaindegree of repair in peripheral skeletal muscles, since the peripheralskeletal muscle contains progenitor cells, which have the capability todivide and replace the peripheral muscle. By isolating those cells froma probe of a thigh muscle, the progenitor cells of skeletal muscle havebeen separated, cultured and re-injected in an animal model (Taylor D.A. et al., “Regenerating functional myocardium: Improved performanceafter skeletal myoblast transplantation,” Nat Med 1998; 4(8):929-33;Scorsin M. et al., “Comparison of the effects of fetal cardiomyocyte andskeletal myoblast transplantation on postinfarction left ventricularfunction,” J Thorac Cardiovasc Surg 2000; 119:1169-75), and morerecently in some patients also.

The application of these cultured cells also been attempted by injectionwith small needles following an opening of the subject's chest and thepericardial sac. While in the model of kiyo-infarction, in which onlythe myocardial cells die but the blood supply through the vascularsystem is not limited, the injection of autologous skeletal myoblastsimproves the myocardial function. The results indicated, however, thatthe engrafted cells retain skeletal muscle characteristic, which meansthey cannot contract at the constant fast rate imposed by thesurrounding cardiac tissue. In addition, no electrical connection existsbetween the graft cells and the host tissue, and it is assumed thattheir contribution to improve contractile performance probably resultedfrom the mechanical ability of the engrafted contractile tissue torespond to stretch activation by contraction.

Considering the experience with latissimus dorsi muscle grafting—aprocedure called dynamic cardiomyoblasty—, the disappointing resultswith the possible use of skeletal muscle as a myocardial substituteindicate that the long term different muscle characteristics of skeletalmuscles do not match the need of a constantly pumping myocardial cell.Therefore, the best these cells might achieve would be to improve thequality of the scar of the ischemic myocardium, but not activelycontribute to a contraction of this area in the long term.

SUMMARY OF THE INVENTION

The present invention is directed to interventional cardiology throughan intraluminal application of cells that have the capability to replacethe necrotic tissue of a failing organ, such as myocardial tissue in thecase MI of the heart, to resume the myocardial function and thereforeimprove the pumping performance of the myocardium. The procedure isoriented on the clinical practice of interventional cardiology followingthe principle that only those approaches that are both (a) relativelyeasy to perform, with little or no risk to the patient but a potentiallyhigh benefit, and (b) highly cost effective, are likely to be routinelyapplied in everyday medicine.

An important aspect of the invention is that the cells to be used in theintraluminal or transluminal application preferably are autologous adultstem cells, which are derived from the same patient that has sufferedthe infarction. The cells are harvested and separated before injection,from the same individual (autologous transplantation). In a case offailing tissue of the myocardium, these cells are then injected into thecoronary artery that caused the infarction.

The approach taken according to the invention recognizes that the stemcells need a certain contact time to adhere and migrate from thevascular bed into the infarcted myocardial area. In contrast to previousapproaches, in which patches or applications through needles into theinfarcted area have been considered, the approach of the inventionhypothesizes that the most effective way to deliver the cells to theinfarcted area is through the vascular tree of coronary arteries,arterioles and capillaries that supply the infarcted area. An occlusionballoon of an over the wire type catheter is inflated at the site of theprimary infarction, after the vessel has been re-canalized and the bloodflow reconstituted.

Importantly, while the blood flow is still blocked, the stem cells aresupplied by slow application through the balloon catheter over arelatively short period of time, on the order of 15 minutes, forexample. That is, the stem cells are injected through the inner lumen ofthe catheter while the balloon is inflated, and therefore, no washoutoccurs. It is believed by applicant that this intracoronary ortranscoronary application of cells during a period that perfusion isceased is critical to enabling the cells to successfully attach to thevessel or myocardial wall. And further, to overcome more actively theendothelial barrier following the increased pressure in the vascularbed, which is attributable to the fact that the retrograde blood flow islimited through the inflated balloon catheter.

The principles of the invention are not limited to cellular repair ofdamaged or failing myocardial tissue, but may be applied in processesfor repair of tissue of various organs of the body, including the brain,liver, kidney or pancreas, for example.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and still further aims, objectives, features, aspects andattendant advantages of the present invention will become apparent tothose skilled in the art from the following detailed description of abest mode presently contemplated of practicing the invention byreference to certain preferred methods of application thereof, taken inconjunction with the accompanying figures of drawing, in which:

FIG. 1 is a transparent front view of a patient showing exemplarylocations of obtaining autologous adult stem cells from the patient, andof injecting the harvested stem cells into the cardiovascular system andthrough a balloon catheter for introduction at the site of myocardialtissue damage to be repaired; and

FIG. 2 is a detail view of the injection of cells at the designatedsite.

DETAILED DESCRIPTION OF THE PRESENTLY CONTEMPLATED BEST MODE OFPRACTICING THE INVENTION

In an exemplary process that applies the principles of the presentinvention to repair of myocardial tissue, the ischemically injuredcardiac tissue is subjected to invasion by stem cells, preferably adultstem cells, with subsequent differentiation into beating cardiomyocyteswhich are mechanically and electrically linked to adjacent healthy hostmyocardium, thereby resembling newly formed and functionally activemyocardium.

Until recently it had been hypothesized by most researchers that adultstem cells are tissue specific. It was thought that a certain stemcell-like population exists in every organ and is capable ofdifferentiation into this certain tissue with exceptions to this ruleregarding repair in heart and brain. Relatively recent studies haveindicated an underestimated potential of these cells. It has been shownthat murine and human neural stem cells (NSC) give rise to skeletalmuscle after local injection (see, for example, Galli R et al.,“Skeletal myogenic potential of human and mouse neural stem cells,” NatNeurosci 2000; 3:986-991). Bone marrow stem cells have also been shownto replace heart tissue (cardiomyocytes, endothelium and vascular smoothmuscle cells) after injection into lethally irradiated mice with amyocardial infarction (see Jackson K. A. et al., “Regeneration ofischemic cardiac muscle and vascular endothelium by adult stem cells,” JClin Invest 2001;107(11):11395-402). The tissue damage in generalappears to transmit signals which direct multi-potential stem cells tothe site of destruction, and these precursors undergo a multi-stepprocess of migration and differentiation at the organ site to replacedamaged cells in form and function.

Experiments with cultured fetal cardiac myocytes or neonatal myocytesimpose limitations owing to their heterologous nature and their possibleinduction of an immuno response necessitating an immuno-suppressivetherapy. Complications and risks associated with an immuno-suppressanttherapy are an increased susceptibility to infection and the possibledevelopment of malignancies. In addition, it is speculated that only afew patients would be willing to undergo a long term immuno-suppressivetherapy with all its negative side effects.

An alternative approach by Prockop suggests that marrow stromal cellsact as stem cells for non hematopoetic tissue and are capable todifferentiate into various types of cells including bone, muscle, fat,hyaline cartilage and myocytes (Prockop D. J. et al., “Marrow stromalcells for non hematopoetic stem tissues,” Science 1997; 276:71-74).

Some recent findings have stimulated interest in adult cardiomyocytes. Areport in Nature describes the ability to inject adult bone marrow stemcells from transgenic mice into the border of infarcted myocardialtissue (Orlic D. et al., “Bone marrow cells regenerate infarctedmyocardium,” Nature 2001; 410:701-5). According to this report, theseadult stem cells are capable of differentiation into cardiomyoblasts,smooth muscle cells and endothelial cells after injection. The infarctedmyocardium implied that the transplanted cells responded to signals fromthe injured myocardium which promoted their migration, proliferation anddifferentiation within the necrotic area of the ventricular wall.

One is then left to consider the most effective techniques to obtainadult stem cells. The classical way to recover stem cells is a bonemarrow tap. The bone marrow contains a wide variety of hematopoetic andmesenchymal stem cells in addition to the T-lymphocytes, macrophages,granulocytes and erythrocytes. By incubation with monoclonal antibodiesspecific for the respective cell lineages and by sorting and removingwith a biomagnet after incubation with magnetic beads and cell sortingwith FACS (fluoroscopy activated cell sorting), a highly enriched cellline of bone marrow derived stem cells can be insulated, cultured andgrown.

Aside from the classical approach of a bone marrow tap, a recent reportstates that cells from human adipose tissue contain a large degree ofmesenchymal stem cells capable of differentiating into different tissuesin the presence of lineage specific induction factors includingdifferentiation into myogenic cells (see Zuk P. A. et al., “Multilineagecells from human adipose tissue: Implications for cell-based therapies,”Tiss Engin 2001; 7(2):211-28). The interesting approach in this researchis that out of a lipoaspirate of 300 cm³ from the subcutanous tissue, anaverage of 2-6×10⁸ cells can be recovered. Even if one assumes thatafter processing of this liposuction tissue and separation and isolationof the mesenchymal stem cells, only 10% of these stem cells might beleft for culture, the remaining approximately 10⁷ (10 million) cellswould be quite sufficient to be used for the intraluminal ortransluminal transplantation process of the present invention.

A benefit of this latter approach could be that culture and passaging ofthe stem cells might be avoided. This is of special importance, since inthe early phases of myocardial infarction there is a high activity ofinflammatory cytokines which promote adhesion, migration andproliferation of the stem cells. In addition, as long as there is noscar core tissue it is much easier for these cells to migrate into thewhole area of myocardial infarction and resume the cardiac function.

Recently, embryonic stem cells have been the subject of intensivediscussion, particularly their pluripotency to differentiate into a vastrange of tissues and organs of the human body that are in need forrepair. The discussion has included the potential use of such stem cellsfor replacement of insulin producing cells as well as embryonic stemcells that can differentiate into cells with structural and functionalproperties of cardiomyocytes. This is described in the August 2001 issueof the Journal of Clinical Investigation (Kehat I. et al., “Humanembryonic stem cells can differentiate into myocytes with structural andfunctional properties of cardiomyocytes,” J Clin Invest 2001;108:407-14). Earlier, the proliferation of embryonic stem cells had beenvery elegantly described in principle by Field and co-workers (Klug M.G. et al., “Genetically selected cardiomyocytes from differentiatingembryonic stem cells form stable intracardiac grafts,” J Clin Invest1996; 98(1):216-24). In their attempt, the latter group succeeded inplating a cell line following a fusion gene consisting of thea—cardiac-myocyte-heavy-chain-promoter and the c-DNA encodingaminoglycosidephosphotransferase that was stably transfected intopluripotent embryonic stem cells. The resulting cell lines weredifferentiated in vitro and subjected to a G418 selection. By thismeans, the selected cardiomyocyte cultures were 99.6% pure and highlydifferentiated.

It is important to consider not only the engraftment of pluripotentembryonic stem cells into a failing organ, but also the possibility ofresulting tumor formation. Therefore, the pluripotent embryonic stemcells need to be cultured in an undifferentiated status, transfected viaelectroporation and grown in differentiated cultures. The interestingapproach in this work is the high yield of selected embryonic stem cellderived cardiomyocytes which, with simple genetic manipulation, can beused to produce pure cultures of cardiomyocytes. The authors assumedthat this elective approach could be applicable to all stem cell derivedcell lineages. It has also been reported by others that isolation ofprimate embryonic stem cells with cardiogenic differentiation isfeasible (Thomson J. A. et al., “Isolation of a primate embryonic stemcell,” Proc Natl Acad Sci USA 1995; 92:7844-48).

In addition, it was recently reported that human cardiomyocytes can begenerated from marrow stromal cells in vitro as well, but with a lowyield of differentiated myocytes (Makino S. et al., “Cardiomyocytes canbe generated from marrow stromal cells in vitro,” J Clin Invest 1999;103:697-705). And as noted above, the 1997 Prockop report in Sciencedescribes another line of cardiomyocytes generated from marrow stromalcells in vitro. This cardiomyogenic cell line was derived from murinebone marrow stromal cells that were immortalized and treated with5-azacytidine. By mechanically separating spontaneously beating cells, acell line was isolated that resembled a structure of fetal ventricularcardiomyocytes expressing iso-forms of contractile protein genes such asalpha cardiomyocyte heavy chain, -light chain, a-actin, Nkx2.5-Csx,GATA-4, tef-1, MEF-2a und MEF-2D.

While these embryonic stem cells provide optimism for the future thatcardiomyocytes derived from embryonic cells might fulfill therequirements of cells that can (a) be passaged indefinitely in culture,(b) be recovered from frozen stocks and are readily available if apatient with a myocardial infarction comes to the cath lab, (c) retaintheir differentiated cardiomyocyte phenotype and (d) maintaincontractile activity with minimum or no immunogenity, further basicresearch is needed on those cells before they can be applied in theanimal model. It is likely that a primate model of infarction and thetransplantation of primate embryonic stem cell derived cardiomyocytesmay be needed as the final proof of principle before a human study mightbe conducted.

Presently, for ethical, immunological and feasibility reasons theapplicant herein submits that transplantation of autologous adult stemcells (derived from the same individual that suffers the infarction) isthe most straightforward and practical approach to repair failingmyocardium. The process of the invention promotes invasion ofischemically injured cardiac tissue by stem cells that firmly attach andsubsequently undergo differentiation into beating cardiomyocytes thatare mechanically and electrically linked to adjacent healthy hostmyocardium. Adhesion of the injected stem cells and their migrationbeyond the endothelial barrier may be confirmed by observation afterseveral days of frozen sections using light microscopy and,subsequently, electron microscopy. For evidence of the transition ofstem cells into cardiomyocytes, markers are introduced into the stemcells before they are reinjected into the myocardial tissue to berepaired.

One approach might be to transplant male cells carrying the Y-chromosomeinto a female organism, but at least two factors weigh against this. Itcould lead to immunologic problems because of the different cellsurfaces carried by the recipient and the donor (heterologoustransplant), a potential reason that some studies are not able to show asuccessful heterologous cell transplantation. Even more importantly, apredominance of inflammatory cells exists at the site of myocardialinjury, which leads to an immediate recognition of foreign cell surfaceproteins with consequent elimination of the cells. Use of autologousstem cells would not carry this immunologic risk of cell destruction,although some difficulty is encountered in prior introduction of geneticor protein markers into those cells.

To overcome this difficulty, a green fluorescence protein (GFP) is usedas a marker, with introduction into the stem cell genome by liposomalgene transfer. Cells can then be identified after transplantation byfluorescence microscopy. As part of the procedure, stem cells are alsomarked by 3H-Thymidin, a radioactive labeled part of DNA. All stem cellsundergoing DNA replication for mitosis will introduce 3H-Thymidin intotheir genome, and thus can be detected afterwards by gamma count. Onelimitation of this process is the fact that radioactivity (per volume)declines with each subsequent cell division (albeit initial total aradioactivity stays constant). Nevertheless, this marker aids indeveloping a gross estimate of the amount of cells in a certain organ ortissue (e.g., heart, spleen, liver etc.).

Reference will now be made to the accompanying Figures of drawing indescribing an exemplary process. It should be noted at the outset thatthe Figures are not intended to be to scale, nor to do more than serveas a visual aid to the description. The autologous adult stem cells(more broadly, myogenic cells) are harvested in one of the known waysgenerally described above, such as by bone marrow tap or from adiposetissue, for cultivation. In an exemplary technique, and referring toFIG. 1, subcutaneous adipose tissue 20 is obtained from a liposuctionprocedure on the patient 1 during local anesthesia. In this procedure, ahollow canule or needle 21 is introduced into the subcutaneous spacethrough a small (approximately 1 cm) cut. By attaching gentle suction bya syringe 22 and moving the canule through the adipose compartment, fattissue is mechanically disrupted and following the solution of normalsaline and a vasoconstrictor epinephrine, a lipoaspirate of 300 cc. isrecovered (retrieved) within the syringe. The lipoaspirate is processedimmediately according to established methods, washed extensively inphosphate buffered saline (PBS) solution and digested with 0.075%collagenase. The enzyme activity is neutralized with Dulbecco's modifiedeagle medium (DMEM) containing 10% FBS (fetal bovine serum) and,following a centrifugation at 1200 G for 10 minutes, a high densitycellular pellet is obtained. Following filtration through anappropriately tight Nylon mesh in order to remove cellular debris, thecells are then incubated overnight in a control medium of DMEM, FBS,containing an antibiotic, antimycotic solution. After the firmattachment of the stem cells to the plate, they are washed extensivelywith PBS solution to remove residual non-adherent red blood cells.Further cellular separation is conducted by separation with monoclonalantibodies coated on magnetic beads. Injection of the lipoaspirate ispreferably performed as early as possible after an infarction (andre-canalization, where that procedure is performed to open a blockedartery that caused the infarction).

Referring to FIG. 2 as well as to FIG. 1, the recovered autologous adultstem cells are transplanted in the donor patient by intracoronary ortranscoronary application for myocardial repair. This process isperformed by first introducing a balloon catheter 11 into thecardiovascular system at the patient's groin 3 using an introducer 4,and through a guiding catheter 5 over a guide wire 18 into the aorta 6and the orifice 7 of a coronary artery 8 of the heart 2 at or in thevicinity of the site where failed tissue owing to an infarction is to berepaired. The failed tissue is supplied with blood through artery 8 andits distal branches 9 and 10. The cells are then injected through theinner (central) lumen 12 of the balloon catheter 11 by means of a motordriven constant speed injection syringe 16 and connecting catheter 17 toentry point of the central lumen at the proximal end of catheter 11. Theexit point of the central lumen 12 is at the distal end of catheter 11which has been advanced into the coronary artery 8 in proximity to thesite of the desired repair. The cells 15 are thereby delivered to thissite by means of slow infusion over 15-30 minutes, for example.

A problem encountered in attempting to do this resides in the fact thatnormally anything inside the blood vessel, including these cells, isseparated from the parenchymatous organ or the tissue outside thevessel. In principle, blood flows through the larger arteries into thesmaller arteries, into the arterials, into the capillaries, and theninto the venous system back into the systemic circulation. Normally, thecells would be prevented from contacting the tissue to be repairedbecause of the endothelial lining and layer of the vessel that protectsthe tissue. However, under certain circumstances this barrier isovercome, and the cells can attach to the inside of the vessel, migrateand proliferate in the adjacent tissue. These circumstances arefacilitated in the case of an acute myocardial infarction, and theincreased pressure in the injection system promotes the injected cellsto overcome the barrier.

The endothelial ischemic damage owing to the infarction allows whiteblood cells, especially granulocytes and macrophages, to attach viaintegrins to the endothelial layer. The endothelial layer itself isdissolved in places by the release of hydrogen peroxide (H₂O₂) whichoriginates from the granulocytes. This mechanism produces gaps in theendothelial layer that allow the stem cells to dock to the endothelialintegrins and also to migrate through these gaps into the tissue to berepaired. An adjacent factor that enables the stem cells to migrate intothe organ tissue is referred to as a stem cell factor that acts as achemo-attractant to the cells.

One is then still faced with the problems of allowing enough of therepair cells to migrate into contact with the failing tissue and ofachieving a high number of transplanted cells in the tissue. This is theprincipal reason for using a balloon catheter 11 or some other mechanismthat will allow the physician (operator) to selectively block theantegrade blood flow and the retrograde stem cell flow. A ballooncatheter is preferred because it is a well known, often used andreliable device for introduction to a predetermined site in a vesselsuch as a coronary artery, to be used for angioplasty for example. Inthe process of the invention, the balloon 14 of catheter 11 is inflatedwith biocompatible fluid through a separate lumen 13 of catheter 11 toocclude coronary artery 8 and its distal branches 9 and 10, therebycausing perfusion through the vessel to cease. Inflation of the balloonmay be commenced immediately before or at the time of injection of thestem cells through the inner lumen of the catheter, and is maintainedthroughout the period of injection. This enables the desired largenumber of adhesions of the cells 15 to the failing tissue to beachieved, because the absence of blood flow at the critical site of thistissue to be repaired has several advantageous effects. It prevents whatwould otherwise result in a retrograde loss of injected cells, aninability to increase the pressure at the injection site to overcome theendothelial barrier and to force the cells through the gap, and anantegrade dilution with blood flow of the cells being injected to thatlocation through the catheter 11.

The blockage is maintained for a relatively short period of time,preferably on the order of fifteen minutes, and in any event sufficientto allow a high concentration and considerable number of cellattachments to the tissue at the designated site, that will tend toguarantee a successful repair. This repair will extend as well to anyfailing tissue that may result from the blockage itself. In the case ofa slow infusion of the cells, the period of blockage is maintainedlonger by steady inflation of the balloon over the injection period, sayup to about 30 minutes, for enhancement of contact and adherence ofabmSC-P to the endothelium. The balloon is deflated, and the ballooncatheter is removed from the patient after the procedure.

Previously reported studies have invariably employed a surgical approachfor the application of the cells to be transplanted. Even if 5 to 10sites of injection are performed with small needles, the complete inner,medial and outer layers of the myocardium are never covered. The conceptof the present invention to use the natural distribution tree of thearterioles and the capillaries is a more elegant solution, provided that(1) the transplanted cells can overcome the endothelial barrier andmigrate into the tissue, (2) interventional cardiology means can restoreblood flow into the infarcted area again, and (3) the cells have enoughtime to overcome the endothelial barrier.

In clinical practice there is a 96% success rate with interventionalcardiology to restore blood flow following an acute myocardialinfarction after an occlusion of a coronary artery. The experience thatvenously injected stem cells can be found in the myocardium, and theknowledge that in an acute myocardial infarction the endothelial barrieris considerably damaged (partly due to the H₂O₂ release of adheringneutrophilic cells), lead to the conclusion that a local injection intothe infarcted area with an occlusive balloon to prevent a washout of thecells is the most desirable approach. The applicant herein has performedstudies in the past with a technique called ‘BOILER’-lysis, insituations where older venous bypass grafts are occluded by a thrombusthat has grown over a prolonged period of time. It was observed that anacute injection of a thrombolytic agent rarely dissolved these oldthrombi. But after an over the wire balloon catheter was inserted intothe occluded graft, a prolonged application of a thrombolytic substancesuch as urokinase was successful in achieving thrombolysis. The agent isinjected at the tip of the balloon catheter, and is forced antegradelyinto the thrombus. The inflated balloon prevents a washout by the normalcoronary circulation and allows the injection by a motor pump at adefined volume per time. For stem cell therapy and repair as performedby the method of the present invention, an injection over a period of 15to 30 minutes is feasible and gives the cells sufficient contact time toadhere to the surface of the damaged endothelium.

The invention is not limited to cellular repair of damaged myocardialtissue. Rather, the process by which the repair is performed may beapplied to the brain in the case of a patient having suffered a cerebralinfarction. Previous studied have indicated that stem cells have thecapacity to replace neural cells in the brain and therefore overturn theconsequences of an acute vascular stroke. In this case, the injectioncatheter is advanced to the site of the damaged tissue through anappropriate arterial path into the applicable region of the patient'sbrain. Blockage of blood flow in this case would add a period (e.g., 15minutes) of limited blood supply but would enable the cells to overcomethe endothelial barrier.

Other possible body organs having damaged tissue to be repaired by theprocess of the invention include the pancreas, the liver, and thekidneys. The pancreas has a duct through which pancreatic enzymes aredelivered into the intestines, and which can be accessed in a retrogrademanner by endoscopic retrograde choledocho-pancreaticography (ERCP).Failing tissue in the case of a diabetic patient means that thepancreatic cells therein no longer produce sufficient insulin for thepatient's needs. By means of a small fiberglass instrument a smallballoon catheter may be introduced into this duct, and the ballooninflated to occlude the duct during delivery of stem cells through thecatheter's inner lumen to the site of the damaged tissue, so as toprevent the injected cells from being washed out into the intestines andthereby enhance large scale adhesions and penetration of the cells tothe target tissue.

An analogous procedure is used for repair of damaged tissue of theliver, through the bile duct system. Here also, it is important toovercome the barrier of the normal bile duct with pressure that can begenerated only if the balloon is inflated while the cells are slowlyinjected. The pressure distally of the injection site increases as moreand more cells are injected. Repair of failing tissue in the kidney(s)may be repaired by an analogous procedure in the case of a renalinfarction.

According to another aspect of the invention, a procedure is used toopen up the blood circulation in an ischemic organ and, additionally, toinject stem cells for repair of tissue damage in the organ occasioned bythe prior blockage. In a myocardial infarction, for example, only aportion of the myocardial cells that had been ischemic will survive,depending on the time period before which restoration of the blood flowis achieved. A typical procedure is to perform a balloon angioplasty ofthe blocked artery, followed by implanting a stent at the site of thelesion. But currently, even in the case of optimal treatment some 40% ofthe affected cells will die. According to this aspect of the invention,within a predetermined brief period after opening the ischemic organ tocirculation of blood flow therethrough, autologous adult stem cells areinjected into the organ proximate the site of the target tissue forrepair thereof. It is imperative that the transplantation of stem cellsbe performed as soon as possible after re-canalization of the organ, soas to take advantage of the profusion of inflammatory cytokines thataccompanies an infarction, which promote the adhesion, migration andproliferation of the stem cells in the tissue. Occlusion of blood flowis desirable here to take advantage of this earlier inflammation.

It should also be noted that while stem cells, particularly autologousadult stem cells, are presently preferred for use in the methods of theinvention, it is possible that certain modified embryonic stem cells orprogenitor cells might be substituted in performing the procedure.

Although a presently contemplated best mode of practicing the inventionhas been disclosed by reference to certain preferred methods, it will beapparent to those skilled in the art from a consideration of theforegoing description that variations and modifications may be madewithout departing from the spirit and scope of the invention.Accordingly, it is intended that the invention shall be limited only bythe appended claims and the rules and principles of applicable law.

What is claimed is:
 1. A process for repairing tissue of a patient'sheart, which comprises delivering stem cells to the site of the tissueto be repaired in a region damaged by myocardial disease through a bloodvessel leading to said site, occluding said vessel during cell deliveryso as to increase the concentration of the delivered stem cells at saidsite, and advancing a catheter through said blood vessel for deliveringsaid stem cells to said site.
 2. The process of claim 1, furtherincluding positioning a guide wire through a portion of the patient'scardiovascular system into said vessel prior to advancing said catheterto said site, and thereafter advancing said catheter over said guidewire for over-the-wire guidance of the distal end of the catheter tosaid site.
 3. The process of claim 1, including employing a ballooncatheter as said catheter, and performing said occlusion of said bloodvessel by inflating the balloon of said catheter for a time intervalprescribed to increase the number of cells delivered to said site.
 4. Aprocess for repairing tissue of an organ in a patient's body, whichcomprises delivering cells that have the capability to replace tissue ofa failing organ to the site of the tissue to be repaired, by anintraluminal application through a blood vessel of said site, andoccluding said blood vessel proximal to the location of cell entrytherein via said intraluminal application during at least a portion ofthe duration of said cell delivery to increase the concentration ofdelivered cells at said site, wherein said organ is the patient's liver,and said blood vessel leads to the site of liver tissue to be repaired.5. A process for repairing tissue of an organ in a patient's body, whichcomprises delivering cells that have the capability to replace tissue ofa failing organ to the site of the tissue to be repaired, by anintraluminal application through a blood vessel of said site, andoccluding said blood vessel proximal to the location of cell entrytherein via said intraluminal application during at least a portion ofthe duration of said cell delivery to increase the concentration ofdelivered cells at said site, wherein said organ is the patient's heart,and said blood vessel leads to the site of heart tissue to be repaired.6. The process of claim 5, including advancing a catheter through saidblood vessel for delivering the cells to said site.
 7. The process ofclaim 6, including introducing a guide wire through said blood vessel tosaid site, and thereafter performing said advancing of the catheter forover-the-wire guidance of the distal end of the catheter along saidguide wire to said site.
 8. The process of claim 6, wherein saidcatheter is a balloon catheter to enable said occlusion of said bloodvessel by inflating the balloon of said catheter for a time intervalprescribed to increase the number of cells delivered to said site. 9.The process of claim 5, including harvesting autologous adult cells fromthe patient's own body, as the source of said cells to be delivered tosaid site.
 10. The process of claim 9, including harvesting said cellsfrom the patient's bone marrow.
 11. The process of claim 9, includingharvesting said cells from the patient's adipose tissue.
 12. The processof claim 9, wherein said cells originate from lipoaspirate.
 13. Theprocess of claim 9, including harvesting said cells from the patient'sown body within a sufficiently short time prior to said delivery toincrease the likelihood of successful tissue repair.
 14. A method ofrepairing tissue of an organ in a patient's body, which comprisesdelivering cells that have the capability to replace tissue of a failingorgan to the site of the tissue to be repaired, by an intraluminalapplication through a duct that normally supports secretion flow fromsaid organ or related gland and leads to said site, including advancinga catheter through said duct for delivering said cells to said site, andoccluding said duct proximal to the location of said intraluminalapplication during said cell delivery to increase the concentration ofdelivered cells at said site, wherein said organ is the patient's liver,said duct is the bile duct, and said catheter is advanced in aretrograde manner through the bile duct.
 15. The method of claim 14,including harvesting autologous adult cells from the patient's own body,as the source of said cells to be delivered to said site.
 16. The methodof claim 15, including harvesting said cells from the patient's bonemarrow.
 17. The method of claim 15, including harvesting said cells fromthe patient's adipose tissue.
 18. The method of claim 15, wherein saidcells originate from lipoaspirate.
 19. The method of claim 15, includingharvesting said cells from the patient's own body within a sufficientlyshort time prior to said delivery to increase the likelihood ofsuccessful tissue repair.